Journal: iScience
Article Title: Stenotrophomonas maltophilia provokes NEU1-mediated release of a flagellin-binding decoy receptor that protects against lethal infection
doi: 10.1016/j.isci.2024.110866
Figure Lengend Snippet: Sm flagellin binds to the MUC1-ED (A) HEK293T cells were transfected with the pcDNA3.1 empty vector, or plasmids encoding for TLR5 (pTLR5) or MUC1 (pMUC1), the cells were lysed and the lysates processed for TLR5 (lanes 1, 2) or MUC1 (lanes 3, 4) immunoblotting. To control for protein loading and transfer, the blots were stripped and reprobed (IB∗) for β-tubulin. (B) HEK293T cells overexpressing TLR5 or MUC1, or empty vector-transfected cells, were incubated with Sm and adherent bacteria quantified. Vertical bars represent mean ± SEM CFUs/well. (C) A549 cells were transfected with pcDNA3.1 or pMUC1 and binding of increasing concentrations of the Alexa Fluor 594-labeled Sm FliC1,2,3 flagellin mixture determined. Data points represent mean ± SEM bound flagellin in molecules/EC. (D) Scatchard analysis of binding data in (C). The linear regression equation and R 2 value are indicated adjacent to each line. (E and F) HEK293T cells were infected with Ad-GFP or Ad-MUC1, and A549 cells were transfected with MUC1-targeting or control siRNAs, and both cell types were cultured for 48 h. (E) Ad-GFP- and Ad-MUC1-infected cells (lanes 1, 2) and MUC1 siRNA- and control siRNA-transfected cells (lanes 3, 4) were lysed and the lysates processed for MUC1 immunoblotting. (F) Infected or transfected cells were incubated with Sm and cell-bound bacteria quantified. Vertical bars represent mean ± SEM Sm adhesion in CFUs/well. (G) A549 cells were transfected with MUC1-targeting or control siRNAs, cultured for 48 h, and binding of increasing concentrations of the Alexa Fluor 595-labeled Sm FliC1,2,3 flagellin mixture determined. Data points represents mean ± SEM bound flagellin in molecules/EC. (H) Scatchard analysis of binding data in (G). (I–K) A549 cells and A549 cells transfected with MUC1-targeting or control siRNAs were incubated for 48 h and then stimulated for 1 h with 10 ng of the Sm FliC1,2,3 flagellin mixture or PBS. (I) Non-transfected, PBS-treated (lane 1), control siRNA-transfected, flagellin-stimulated (lane 2), and MUC1 siRNA-transfected, flagellin-stimulated (lane 3) cells were lysed, and the lysates processed for pERK1/2 immunoblotting. To control for protein loading and transfer, the blots were stripped and reprobed for total ERK2. The ∼44.0 kDa pERK1 (upper) and ∼42.0 kDa pERK2 (lower) bands, as well as the total ERK2 band, are indicated by arrowheads on the right. (J) Densitometric analysis of pERK1/2 normalized to total ERK2 displayed in (I). Vertical bars represent mean ± SEM normalized pERK1/2 signal. (K) Supernatants from the cells in (I) were processed for IL-8 levels and normalized to total EC protein. Vertical bars represent mean ± SEM normalized IL-8 concentrations in pg/mg EC protein. ∗, increased Sm adhesion (B, F), flagellin binding (C), ERK activation (J), or normalized IL-8 release (K) compared to pcDNA3.1, pTLR5, Ad-GFP, control siRNA, or PBS controls at p < 0.05. ∗∗, decreased Sm adhesion (F), flagellin binding (G), ERK activation (J), or normalized IL-8 release (K) compared to the siRNA controls at p < 0.05. n.s., not significant. Statistical comparisons were made using the Student’s t test. The results are representative of 3 or 6 independent experiments.
Article Snippet: Mouse anti-human TLR5 , Santa Cruz Biotechnology , Cat# sc-52963; RRID: AB_793184.
Techniques: Transfection, Plasmid Preparation, Western Blot, Control, Incubation, Bacteria, Binding Assay, Labeling, Infection, Cell Culture, Activation Assay
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